Elektrophysiologische Charakterisierung Insulin sezernierender beta-Zellen in Gewebeschnitten des Pankreas

In der vorliegenden Studie wurde eine Präparation pankreatischer Gewebeschnitte etabliert, um unter Bedingungen nahe in vivo, die Charakterisierung Insulin sezernierender β-Zellen, zu ermöglichen. Das Arbeitsverfahren zur Herstellung von Gewebeschnitten des Pankreas ist schnell und leicht reproduzierbar. Die Gewebeschnitte waren für mindestens einen Tag lebensfähig und die grobe Morphologie war gut erhalten. Durch Messung der Insulinfreisetzung nach Applikation hoher Glukose Konzentrationen wurde die Funktionalität der β-Zellen in den Gewebeschnitten bestätigt. Die Gewebeschnitt Präparation des Pankreas ermöglichte die Durchführung elektrophysiologischer Experimente an β-Zellen, die sich in Tieferen Lagen der Langerhansschen Insel befanden. Die Charakterisierung der β-Zellen in Gewebeschnitten enthüllte mehrere verschiedene Eigenschaften im Vergleich zu den Präparationen, die bisher verwendet wurden. Erstens war die durch Kapazitätsmessungen erhaltene Sekretionsrate der β-Zellen in Gewebeschnitten vergleichbar mit der mit biochemischen Methoden gemessenen Insulinfreisetzung im perfundierten Pankreas. Experimente an kultivierten β-Zellen und β-Zellen in Langerhansschen Inseln zeigen diese Übereinstimmung nicht. Weiterhin wiesen KATP-Kanäle in β-Zellen in Gewebeschnitten, verglichen mit getrennten β-Zellen, eine verringerte Sensitivität gegenüber ATP auf, wodurch die Regulation der KATP-Kanäle durch ATP in ein physiologisches Niveau verschoben wird. Zusätzlich zeigte sich, dass das Auslaufen der KATP-Kanal Aktivität in β-Zellen in Gewebeschnitten beschleunigt war. Aufnahmen elektrischer Aktivität von β-Zellen in Gewebeschnitten, als Antwort auf stimulierende Substanzen, wiesen das charakteristische, oszillierende Muster auf. Jedoch zeigten Studien an Mäusen, die keine Gap Junctions zwischen β-Zellen ausbildeten, dass die elektrische Aktivität in β-Zellen durch den so genannten wash-out nach der Dialyse der Pipettenlösung verschwand. Die elektrische Aktivität aufgenommen von wild Typ Mäusen in der whole-cell Konfiguration reflektierte daher die Aktivität gekoppelter β-Zellen. Weiterhin verhinderte das Vorhandensein von Gap Junctions elektrische Aktivität in einer einzelnen, stimulierten β-Zelle, so lange sich die Mehrheit der gekoppelten Zellen noch in einem Ruhezustand befanden. Dies trägt zur Synchronisierung der Insulinfreisetzung von der Langerhansschen Insel bei. Die Studie stellte Gewebeschnitte des Pankreas als eine aussichtsreiche, weniger invasive Methode zur Untersuchung der β-Zell-Funktion dar. In dieser Präparation zeigten β-Zellen Eigenschaften, die für in vivo Bedingungen angenommen werden. Die Anwendung pankreatischer Gewebeschnitte wird als ein geeignetes System dienen, um weiteren Einblick in die Komplexen Interaktionen zu erhalten, welche die Homöostase der Glukose im Blut kontrollieren

Insulin Release: Shedding Light on a Complex Matter

SNAP25 and syntaxin-1 are core proteins of the exocytosis machinery for insulin granules. Takahashi et al. (2010) show that the distribution and kinetics of the SNAP25/syntaxin-1 complex assembly play a key role in regulating the first and second phase of insulin release

Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology in situ

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ

Oxidative Stress, Antioxidants and Hypertension

As a major cause of morbidity and mortality globally, hypertension remains a serious threat to global public health. Despite the availability of many antihypertensive medications, several hypertensive individuals are resistant to standard treatments, and are unable to control their blood pressure. Regulation of the renin-angiotensin-aldosterone system (RAAS) controlling blood pressure, activation of the immune system triggering inflammation and production of reactive oxygen species, leading to oxidative stress and redox-sensitive signaling, have been implicated in the pathogenesis of hypertension. Thus, besides standard antihypertensive medications, which lower arterial pressure, antioxidant medications were tested to improve antihypertensive treatment. We review and discuss the role of oxidative stress in the pathophysiology of hypertension and the potential use of antioxidants in the management of hypertension and its associated organ damage

Oxidative Stress, Antioxidants and Hypertension

As a major cause of morbidity and mortality globally, hypertension remains a serious threat to global public health. Despite the availability of many antihypertensive medications, several hypertensive individuals are resistant to standard treatments, and are unable to control their blood pressure. Regulation of the renin-angiotensin-aldosterone system (RAAS) controlling blood pressure, activation of the immune system triggering inflammation and production of reactive oxygen species, leading to oxidative stress and redox-sensitive signaling, have been implicated in the pathogenesis of hypertension. Thus, besides standard antihypertensive medications, which lower arterial pressure, antioxidant medications were tested to improve antihypertensive treatment. We review and discuss the role of oxidative stress in the pathophysiology of hypertension and the potential use of antioxidants in the management of hypertension and its associated organ damage

Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology <i>in situ</i>

<div><p>Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells <i>in vitro</i>. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding <i>in situ</i>.</p></div

Acinar cell function during long-term culture of pancreas tissue slices.

<p><i>(A)</i> Amylase release from freshly prepared and cultured pancreas tissue slices after 30 min stimulation with indicated caerulein concentrations. The relative amount of released amylase decreases with culture time. However, amylase release shows a typical bell-shaped curve response to increasing caerulein concentrations at all time points. Amylase release is expressed as percent of total amylase as mean ± SD of 16 slices per time point (n = 16). <i>(B)</i> Traces of Ca²⁺_i/Oregon Green BAPTA-1 fluorescence in acinar cells of pancreas tissue slices at indicated time points after preparation. Stimulation with 10 pmol/L caerulein induced oscillations of Ca²⁺_i/Oregon Green BAPTA-1 fluorescence in acinar cells of slices at all time points. Traces are shown as fluorescence ratio (F/F₀), in which F is fluorescence at any given time and F₀ is pre-stimulatory fluorescence.</p

Effect of long-term pancreas tissue slice culture on endocrine beta cell viability and function.

<p>(<i>A</i>) Longitudinal <i>in situ</i> imaging of beta cell viability and specificity in pancreas tissue slices before and after 7 days culture in standard and optimized conditions. Under standard culture conditions MIP-GFP fluorescence (green) of beta cells was lost after 7 days and the numbers of dead nuclei (magenta) dramatically increased, whereas in optimized conditions beta cells were still detectable by GFP fluorescence after 7 days and the number of dead cells inside the islet remained low. <i>(B)</i> Quantification of Draq7 nuclei (dead cells) within islets in freshly prepared pancreas tissue slices and during culture in standard and optimized conditions. Islet volume in tissue slices was determined by backscatter LSM. Values are expressed as mean ± SD Draq7 labeled nuclei per mm³ islet volume (n = 11 for optimized and n = 12 for standard conditions). <i>(C)</i> Basal (3 mmol/L glucose) and stimulated (16.7 mmol/L glucose) insulin release from pancreas tissue slices at the day of preparation and after 4 and 7 days of culture in optimized conditions. Insulin release is expressed as percent of total insulin as mean ± SD from 8 slices per time point (n = 8).</p